Treatment of amyotrophic lateral sclerosis

ABSTRACT

The present invention relates to the treatment of motoneuron diseases. More particularly the invention relates to the treatment of amyotrophic lateral sclerosis (ALS). It is found that the intracerebroventricular delivery of low amounts of vascular endothelial growth factor into a preclinical ALS animal model induces a significant motor performance and prolongation of survival time of said animals.

FIELD OF THE INVENTION

The present invention relates to the treatment of motoneuron diseases.More particularly the invention relates to the treatment of amyotrophiclateral sclerosis (ALS). It is found that the intracerebroventriculardelivery of low amounts of vascular endothelial growth factor into apre-clinical ALS animal model induces a significant motor performanceand prolongation of survival time of said animals.

INTRODUCTION TO THE INVENTION

Amyotrophic lateral sclerosis (ALS) is a devastating paralyzingdisorder, killing patients within 3 to 5 years after onset¹⁻⁴. Clinicalsymptoms primarily result from progressive degeneration of motoneuronsin the spinal cord and brain stem, largely sparing cognitiveperformance. The disease affects healthy individuals in the midst oftheir life, sporadically in >90% of cases without any family history.Although more males than females are affected before the age of 60years, both genders are similarly affected at older age⁵. With theageing population, increasingly more individuals suffer ALS—the thirdmost common neurodegenerative disorder⁶. Many ALS patients first noticemuscle weakness in their limbs (“limb-onset” ALS). In ˜25% of ALSpatients, motoneurons first degenerate in the motor nuclei of the brainstem (“bulbar-onset” ALS), causing dysarthria, dysphagia and respiratoryproblems. Bulbar-onset ALS patients generally exhibit a faster and moreaggressive disease progression than do limb-onset patients, but thelatter eventually develop bulbar symptoms as well. The precise cause ofmotoneuron degeneration in most cases remains largely enigmatic^(2,4,7).SOD-1 mutations cause motoneuron degeneration in humans and, whenoverexpressed, also in transgenic mice. In fact, SOD-1^(G93A) mice havebecome the gold standard animal model to assess the therapeuticpotential of novel drug candidates⁸. SOD-1^(G93A) rats develop anaggressive form of ALS, but have not been used yet for evaluation ofnovel treatments^(9,10). No approved, effective cure is available yetfor ALS. Riluzole is the only approved drug in some but not allcountries, but it has a marginal benefit on survival, is costly, notfree of side-effects and, importantly, ineffective on bulbar symptoms¹⁶.As ALS results from degeneration of motoneurons, neurotrophic growthfactors, such as brain-derived neurotrophic factor (BDNF), ciliaryneurotrophic factor (CNTF), insulin-like growth factor (IGF)-1,leukemia-inhibitory factor (LIF), cardiotrophin (CT)-1 and hepatocytegrowth factor (HGF) have long been considered as therapeutic candidatesfor ALS. Gene transfer of GDNF but especially of IGF-1, using aretrogradelly axon-transported viral vector, has been shown to prolongsurvival of SOD-1^(G93A) mice^(17,18). Though clinical trials areunderway^(c), the clinical applicability of gene therapy for ALS remainsto be established and concerns about its irreversible nature, risk foradverse chromosomal effects, poor control of transgene expression, andlarge production needs still remain to be overcome. Delivery ofrecombinant neurotrophic growth factors, instead, is therefore anattractive therapeutic strategy, as it offers flexible control of thedose and duration of the administered protein. However, intrathecalinfusion of BDNF¹⁹, intracerebroventricular delivery of GDNF^(a) orsystemic administration of BDNF^(b) or CNTF²⁰ has, to date, not resultedin substantial clinical improvement in ALS patients, except for a 26%slowing of disease progression after IGF-1 delivery in one but not inanother study^(21,22). At least part of the failure can be ascribed tothe short half-life, immunogenicity, dose-dependent dual effect onneuronal survival versus apoptosis, undesired toxicity and limitedability to cross the blood-brain barrier after systemic delivery ofthese proteins²³⁻²⁵. Another possible reason may relate to the fact thatseveral of these factors, even while promoting survival of acutelyinjured motoneurons when exogenously supplied, may not play such acritical role in the endogenous control of adult motoneuron survival ina chronic disease such as ALS. We recently discovered that low VEGFlevels are redundant for motoneuron development but cause adult-onsetALS-like motoneuron degeneration in genetically modified mice(WO0176620) and increase the risk of sporadic and familial ALS in humansas well¹³⁻¹⁵. VEGF is a prototype angiogenic factor, implicated invessel growth in health and disease^(11,12). To avoid immune problemsand systemic side effects, to overcome the limited ability of VEGF tocross the blood-brain barrier, and to achieve maximal VEGF proteinlevels in the spinal cord parenchyma, we developed a strategy, neverpreviously used to examine the therapeutic potential of a recombinantprotein in preclinical ALS studies, i.e. to deliver,intracerebroventricularly, recombinant VEGF for prolonged periods usinga transgenic SOD-1^(G93A) rat model of ALS. Surprisingly, we have foundthat extremely low levels of VEGF not only significantly amelioratesmotoric performance but also prolongs the survival time in a ratpre-clinical ALS model with an unexpected long time. The results showthat low levels of VEGF can slow down the disease progression ofpatients suffering from ALS when administratedintracerebroventricularly. ^(c)world wideweb.hdlighthouse.org/research/genetherapy/updates/0052als.phtml.^(a)worldwide web.mdausa.org/research/ct-alsglia.html.^(b)world wideweb.alsa.org/news/news01280-1.cfm

DETAILED DESCRIPTION OF THE INVENTION

The present invention shows that intracerebroventricular (ICV) deliveryof low amounts of VEGF delays onset, ameliorates motoric performance andprolongs survival of a rat strain suffering an aggressive form of ALS.This is the first study showing a significant therapeutic effect of arecombinant growth factor on the disease characteristics in apreclinical ALS model. Several recombinant growth factors with knownneurotrophic activity, including BDNF, IGF-1, CNTF, LIF and GDNF havebeen evaluated in ALS patients or SOD1^(G93A) mice, but not a singlerecombinant growth factor provided consistently a substantialbenefit^(19-22,35), therefore since the failure of previous clinicaltrials¹⁹⁻²² administration of recombinant growth factors has lost muchof its attraction. As we show here VEGF has direct effects onmotoneurons in vivo. Although VEGF is known to affect various neuronalprocesses in vivo¹⁵, it has never been established whether VEGF exertedits effects directly on neurons or indirectly through other cell typesor via other molecular intermediates. Thus, VEGF has a pleiotropicspectrum of activities, which may have contributed to its remarkabletherapeutic benefit in this invention.

The present invention indicates that VEGF can be used to manufacture amedicament for the treatment of motoneuron disorders and morespecifically for the treatment of amyotrophic lateral sclerosis andamyotrophic lateral sclerosis-like diseases. In a particular embodimentthe VEGF₁₆₅-isoform is used for the manufacture of a medicament for thetreatment of motoneuron disorders and more specifically for thetreatment of amyotrophic lateral sclerosis and amyotrophic lateralsclerosis-like diseases wherein said VEGF is continuously administeredclose to the place of onset.

VEGF₁₆₅ is a 165-amino acid protein which is typically referred to ashuman VEGF (hVEGF). VEGF is expressed in a variety of tissues asmultiple homo-dimeric forms (121, 145, 165, 189, and 206 amino acids permonomer) resulting from alternative RNA splicing. VEGF₁₂₁ is a solublemitogen that does not bind heparin; the longer forms of VEGF bindheparin with progressively higher affinity. The heparin-binding forms ofVEGF can be cleaved in the carboxy terminus by plasmin to release adiffusible form(s) of VEGF. In addition several molecules structurallyrelated to VEGF have also been identified recently, including placentagrowth factor (PIGF), VEGF-B, VEGF-C, VEGF-D and VEGF-E. Ferrara andDavis-Smyth (1997) Endocr. Rev., Ogawa et al. (1998) J. Biological Chem.273:31273-31281; Meyer et al. (1999) EMBO J., 18:363-374.

The terms ‘pharmaceutical composition’ or ‘medicament ’ or ‘use for themanufacture of a medicament to treat’ relate to a composition comprisingVEGF as described above and a pharmaceutically acceptable carrier orexcipient (both terms can be used interchangeably) to treat motoneurondiseases as indicated above. Suitable carriers or excipients known tothe skilled man are saline, Ringer's solution, dextrose solution, Hank'ssolution, fixed oils, ethyl oleate, 5% dextrose in saline, substancesthat enhance isotonicity and chemical stability, buffers andpreservatives. Other suitable carriers include any carrier that does notitself induce the production of antibodies harmful to the individualreceiving the composition such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids and amino acidcopolymers. In a particular embodiment the ‘medicament’ may beadministered by a method close to the place of onset. Indeed, thespatial gradient of VEGF levels and therapeutic benefit indicates that,despite the significant CSF turnover, VEGF is deposited in closeproximity to its injection site. This might offer novel opportunities totailor VEGF therapy to the patients' needs. Indeed, individualssuffering ALS with bulbar onset can benefit more from ICV or intrathecaldelivery of VEGF at the cervical level, while ALS patients with lumbaronset can benefit more from an intrathecal infusion of VEGF at thelumbar level. Generally, the medicament is administered so that VEGF,more particularly VEGF₁₆₅, is given at a dose between 0.01 μg/kg/day and1.5 μg/kg/day, more preferably between 0.05 μg/kg/day and 1 μg/kg/day,most preferably between 0.2 μg/kg/day and 0.8 μg/kg/day. Preferably acontinuous infusion is used and includes the continuous subcutaneousdelivery via an osmotic minipump. In another embodiment VEGF₁₆₅ is usedfor the manufacture of a medicament to treat motoneuron disease whereinsaid VEGF₁₆₅ is continuously administered close to the place of onset ata dose within a range between 0.05 μg/kg/day and 1 μg/kg/day. In anotherembodiment VEGF₁₆₅ is used for the manufacture of a medicament to treatmotoneuron disease wherein said VEGF is continuously administered closeto the place of onset at a dose within a range between 0.2 μg/kg/day and0.8 μg/kg/day.

In another embodiment said close to the onset administration is anintrathecal administration.

In another embodiment said close to the onset administration is anintracerebroventricular administration. It should also be clear that theadministration of VEGF₁₆₅ is continuously administered close to theplace of onset at a dose within a range between 0.01 μg/kg/day-1.4μg/kg/day or 0.01 μg/kg/day-1.3 μg/kg/day or 0.01 μg/kg/day-1.2μg/kg/day or 0.01 μg/kg/day-1.1 μg/kg/day or 0.01 μg/kg/day-1 μg/kg/dayor 0.01 μg/kg/day-0.9 μg/kg/day or 0.01 μg/kg/day-0.8 μg/kg/day or 0.01μg/kg/day-0.7 μg/kg/day or 0.01 μg/kg/day or 0.6 μg/kg/day or 0.01μg/kg/day-0.5 μg/kg/day or 0.01-0.4 μg/kg/day or 0.01 μg/kg/day-0.3μg/kg/day or 0.01 μg/kg/day-0.2 μg/kg/day or 0.01 μg/kg/day-0.1μg/kg/day or 0.01 μg/kg/day-0.09 μg/kg/day or 0.01 μg/kg/day-0.08μg/kg/day or 0.01 μg/kg/day-0.07 μg/kg/day or 0.01 μg/kg/day-0.06μg/kg/day or 0.01 μg/kg/day-0.05 μg/kg/day or 0.01 μg/kg/day-0.04μg/kg/day or 0.01 μg/kg/day-0.03 μg/kg/day. It should also be clear thatthe administration of VEGF₁₆₅ is continuously administered close to theplace of onset at a dose within a range between 0.02 μg/kg/day-1.5μg/kg/day or 0.03 μg/kg/day-1.5 μg/kg/day or 0.04 μg/kg/day/kg/day-1.5μg/kg/day or 0.05 μg/kg/day-1.5 μg/kg/day or 0.06 μg/kg/day-1.5μg/kg/day or 0.07 μg/kg/day-1.5 μg/kg/day or 0.08 μg/kg/day-1.5μg/kg/day or 0.09 μg/kg/day-1.5 μg/kg/day or 0.1 μg/kg/day-1.5 μg/kg/dayor 0.2 μg/kg/day-1.5 μg/kg/day or 0.3 μg/kg/day-1.5 μg/kg/day or 0.4μg/kg/day-1.5 μg/kg/day or 0.5 μg/kg/day-1.5 μg/kg/day or 0.6μg/kg/day-1.5 μg/kg/day or 0.7 μg/kg/day-1.5 μg/kg/day or 0.8μg/kg/day-1.5 μg/kg/day or 0.9 μg/kg/day-1.5 μg/kg/day or 1μg/kg/day-1.5 μg/kg/day or 1.1 μg/kg/day-1.5 μg/kg/day or 1.2μg/kg/day-1.5 μg/kg/day or 1.3 μg/kg/day-1.5 μg/kg/day.

Thus in a particular embodiment the infusion with a compositioncomprising VEGF is intrathecal. Intrathecal administration can forexample be performed by means of surgically implanting a pump andrunning a catheter to the spine.

To clarify the invention the term ‘motoneuron disease’ is explainedbelow. Motoneuron disease is a group of diseases involving thedegeneration of the anterior horn cells, nerves in the central nervoussystem that control muscle activity. This leads to gradual weakening andeventually wasting of the musculature (atrophy). Diseases of themotoneuron are classified according to upper motoneuron (UMN) and/orlower motoneuron (LMN) involvement. Upper motoneurons originate in thebrain, in particular, the motor cortex, and they synapse either directlyor indirectly onto lower motoneurons. Upper motoneurons are moreaccurately referred to as pre-motoneurons, and they are responsible forconveying descending commands for movement. Lower motoneurons aredevisable into two categories: visceral and somatic motoneurons.Visceral motoneurons are autonomic pre-ganglionic neurons that regulatethe activity of ganglionic neurons, which innervate glands, bloodvessels, and smooth muscle. Somatic motoneurons innervate skeletalmuscle and include first, anterior horn cells, which as the nameimplies, are located in the anterior horn of the spinal cord, andsecond, lower motoneurons located in the cranial nerve nuclei.Amyotrophic lateral sclerosis or ALS is the most frequent form(accounting for around 80% of all cases) of motoneuron disorders. ALS isknown as Lou Gehrig's disease, named after the famous Yankee baseballplayer. The initial symptoms of ALS are weakness in the hands and legsand often fasciculation of the affected muscles. Whichever limbs areaffected first, all four limbs are affected eventually. Damage to theupper motoneurons produces muscle weakness, spasticity and hyperactivedeep tendon reflexes. Lower motoneuron damage produces muscle weaknesswith atrophy, fasciculations, flaccidity and decreased deep tendonreflexes. ALS has features of both upper and lower motoneurons of thecranial nerves, therefore symptoms are isolated to the head and neck.Some patients will also display UMN involvement of the cranial nervesand if this is the sole manifestation it is referred to as Pseudobulbarpulsy. Spinal muscular atrophy or progressive muscular atrophy is amotoneuron disease that does not involve the cranial nerves and is dueto lower motoneuron degeneration. Shy-Drager syndrome is characterizedby postural hypotension, incontinence, sweating, muscle rigidity andtremor, and by the loss of neurones from the thoracic nuclei in thespinal cord from which sympathetic fibres originate. Destructive lesionsof the spinal cord result in the loss of anterior horn cells. This isseen in myelomeningocele and in syringomyelia, in which a largefluid-filled cyst forms in the centre of the cervical spinal cord.

The beneficial effect with VEGF relates to the fact that in the presentinvention this recombinant growth factor was delivered continuously anddirectly into the cerebrospinal fluid (CSF)—the availability of thelarger SOD1^(G93A) rat models of ALS was therefore instrumental. Thebiodistribution data show that VEGF, after ICV delivery, is rapidlydiffusing from the CSF into the spinal cord parenchyma and thus capableof reaching the lower spinal motoneurons though we cannot exclude thatsome of the benefit may also have come from an effect on uppermotoneurons. The close correlation between the biodistribution of VEGF(higher in brain stem than in the lumbar spinal cord) and itstherapeutic effect shows that VEGF induced its greatest effect at siteswhere its levels were highest. As VEGF is cleared from the centralnervous system beyond 3 hours (similar as other neurotrophins³⁸),continuous delivery of VEGF likely contributed to the beneficial effect.Other reasons may relate to the general safety profile ofICV-administered VEGF (we could not observe any prominent adverseeffects when using a therapeutic dose, which was 10-fold below thetoxicity threshold) and the lack of an immune response (which might haveotherwise induced neutralizing antibodies). An immune reaction did occurupon systemic delivery of VEGF and, likely, the negligible penetrationof VEGF across the blood-brain barrier would be only overcome byadministering systemically massive—likely toxic—amounts of VEGF.

ICV delivery may seem cumbersome at first sight, but is technicallyfeasible and already operational for other chronic neurologicalindications⁴³. The discomfort of a single surgical intervention toposition the pump may, in fact, greatly outweigh the benefits of thisroute of administration (lack of systemic side effects and immuneresponse; controllable administration). Our previous genetic dataindicate that VEGF affects both sporadic and familial ALS¹⁴. While weonly could use, in the present invention, an animal model of familialALS, there is a good chance that VEGF will be also effective in sporadiccases of ALS. After all, VEGF has survival effects for various types ofneurons, regardless of the sort of stress (hypoxia, excitotoxicity,serum deprivation, mutant SOD1-related toxicity, etc) and is thus anattractive candidate¹⁵. VEGF therapy appears to be safe and welltolerated for protracted time periods, without causing vascular side.Our findings extend previous observations that VEGF is known to havequalitatively distinct biological activities (vascular permeability,angiogenesis) at different concentrations⁵⁷. Moreover, low VEGF levelsonly stimulate angiogenesis in ischemic/injured brain when delivereddirectly into the brain parenchyma, but not into the CSF^(48,58).

EXAMPLES 1. Systemic Versus Intracerebroventricular Delivery ofRecombinant VEGF

We first assessed via which route, i.e. systemic orintracerebroventricular, VEGF would be best administered. Safe andeffective growth factor therapy of ALS requires that administration ofthe protein should not mount an immune response or induce systemicadverse effects, but result in sufficiently high, i.e. therapeutic,levels in the spinal cord parenchyma. Initial studies indicated that allcommercially available recombinant human, rat or murine VEGFpreparations, when administered intraperitoneally to mice at 1 μg everytwo days (a therapeutic dose to stimulate angiogenesis³⁷), caused astrong immune response within 2 weeks. The best results were obtainedwhen using in-house E. coli-produced murine VEGF, but even thispreparation caused an immune response within 4 weeks, thereby precludingchronic systemic delivery of VEGF. In addition, VEGF is hydrophilic andhas a molecular weight of 44 kDa and is thus unlikely to cross theblood-brain or blood-cerebrospinal fluid (CSF) barrier with anefficiency of more than 1% of the injected dose³⁸. To obtain sufficientVEGF levels in the spinal cord parenchyma, excessive amounts of VEGF,likely inducing toxic systemic side effects, would therefore have to beadministered. Furthermore, VEGF is trapped not only by heparansulfate-rich extracellular matrix in tissues but also, in the plasma, bysoluble Flt1 (sFlt1), i.e. the extracellular ligand-binding domain ofVEGF receptor-1 (also termed Flt1)^(39,40). We detected, however, 5-foldhigher soluble Flt1 levels in the serum than in the cerebrospinal fluidin mice (3,800±594 μg/ml versus 810±66 μg/ml; N=3; P<0.05). Thus, alarger fraction of VEGF would be trapped by sFlt1 and heparansulfate-rich matrix, when administered systemically thanintracerebroventricularly. Lastly, although VEGF levels are undetectablein the CSF⁴¹, the choroid plexus is one of the few sites in the body,where VEGF remains constitutively expressed in the adult⁴². Because ofall these reasons, we evaluated whether intracerebroventricular (ICV)delivery of VEGF would offer an alternative approach and optimized thenecessary techniques for long-term ICV delivery of growth factors. As wepreviously determined that the VEGF¹⁶⁴ isoform (the human equivalent isVEGF₁₆₅) exhibits the optimal biological properties to stimulatemotoneuron survival¹³ (WO0176620), we used this isoform in the rest ofour study. Since rats were used for all delivery experiments (seebelow), we cloned and expressed a rat VEGF¹⁶⁴ protein preparation, whichwas >99% pure and endotoxin-free.

2. Biodistribution of VEGF after Intracerebroventricular Delivery

Nothing is known about the pharmacokinetics of VEGF in the centralnervous system after ICV delivery³⁸. It is even unknown whether VEGFadministered into the CSF would be capable of diffusing into the spinalcord parenchyma across the ependymal barrier. We therefore firstdetermined the distribution pattern of ¹²⁵I-VEGF after ICV delivery inhealthy rats. At one hour after a bolus ICV injection, only 12% of theinjected amount of ¹²⁵I-VEGF was still present in the CSF, whereas 70%and 12% were recovered in the parenchyma of the brain and spinal cord,respectively, indicating that ¹²⁵I-VEGF readily diffused from the CSFinto the parenchyma. Thus, similar as for NGF, but unlike BDNF, theependymal layer does not represent a barrier for VEGF to diffuse intothe parenchyme³⁸. When expressing the ¹²⁵I-VEGF levels per gram tissue,¹²⁵I-VEGF levels were highest in the vicinity of the injection site andprogressively declined in a rostro-caudal gradient along the spinalcord: ¹²⁵I-VEGF levels in the bulbar/cervical, thoracal and lumbarspinal cord were 80%, 50% and 16%, respectively, of those in the brain.At 3 hours after injection, only 30% of the injected ¹²⁵I-VEGF waspresent in the brain and spinal cord, while negligible amounts weredetectable after 24 hours, when ¹²⁵I-VEGF was recovered in theexcretions, suggesting that VEGF was cleared into the venous andlymphatic systems, as documented for other growth factors³⁸. Twoconclusions can be drawn from these experiments. First, ICV-deliveredVEGF rapidly diffuses from the CSF to neurons in the parenchyma, but isthen cleared within 24 hours from the CSF. As motoneurons in ALSchronically require survival signals, VEGF should be deliveredcontinuously. Second, after ICV delivery, VEGF is distributed in arostro-caudal gradient—this can have consequences for affectingmotoneuron survival in a similar spatial pattern.

3. Long-Term Intracerebroventricular Delivery of Recombinant VEGF

Although prolonged ICV delivery of GDNF for up to 8 months has beenachieved in humans for other neurodegenerative disorders⁴³, this routehas not been used to evaluate the therapeutic potential of recombinantgrowth factors in preclinical ALS models. To achieve long-termcontinuous and constant delivery of recombinant VEGF, we implantedosmotic minipumps subcutaneously on the backs of the animals andconnected them to a catheter, which we stereotactically positioned inthe lateral ventricle of the brain. While technically feasible tocatheterize the lateral ventricle in mice, the catheters failed toremain fixed in the brain and became detached after several weeks, asthe mice tried to remove them by scratching their head. In addition, thelarge size of the pump, relative to the body size (almost 30% of theirbody size), precluded reliable motor performance measurements. Wetherefore chose to implant pumps, delivering compounds for up to 4weeks, in an SOD1^(G93A) rat ALS model to assess the therapeuticpotential of ICV delivery of recombinant VEGF protein. By replacing thepumps every 4 weeks, we succeeded in reproducibly delivering VEGFprotein for more than 100 days without any adverse effects (see below).The correct position and patency of the catheters was checked in eachrat at the time of dissection. Importantly, VEGF was still biologicallyactive in binding its receptors, VEGF receptor-1 (also termed Flt1) andVEGF receptor-2 (Flk1), after being incorporated into the osmoticminipump in the animals for several weeks. Indeed, when retrieved fromthe pumps after three weeks, 89% and 68% of the residual VEGF in thepump still bound Flt1 and Flk1, respectively, indicating that themajority of the rVEGF¹⁶⁴ stored in the pumps was still active. As VEGFhas never been administered to the central nervous system chronically(the longest duration was one week) and the effects of acute versuschronic administration of VEGF may differ, we carefully assessed whethera particular dose of VEGF would stimulate motoneuron survival withoutcausing excessive blood vessel growth or leakage, even not afterprolonged delivery for several months. Initial experiments revealed thatrats receiving intracerebroventricularly a high dose of VEGF (e.g. 20μg/kg/day) all died after a few days. At 2 μg/kg/day, 66% of the ratsbecame ill after 3 to 4 weeks. While they were not paralyzed (theanimals could still walk normally when pushed), they were generallyapathic. Macroscopic inspection and histological analysis of the brainand spinal cord revealed additional vessel growth, ventriculardilatation, and redness and edema of the brain and spinal cord. A doserange between 0.2 μg/kg/day and 0.6 μg/kg/day was well tolerated by therats, without inducing edema, leakage or excess vessel growth—even whenadministered chronically for more than 100 days. At this dose, VEGFlevels in the CSF remained undetectable. SOD1^(G93A/LSd) rats(SOD1^(G93A) rats with low SOD1^(G93A) expression on Sprague-Dawleybackground; see example 4) were treated with 0.6 μg VEGF/kg/day. At thisdose, VEGF levels in the CSF remained undetectable, presumably becauseinfused VEGF rapidly diffused into the spinal parenchyma. This isimportant, as only detectable VEGF levels have been associated withpathology⁴¹. We therefore used a dose of 0.6 μg VEGF/kg/day to treatSOD1^(G93A) rats. This dose is, even after correction for the relativedistribution volume of the entire body versus the brain/spinal cord,still 5-fold lower than a dose used for therapeutic angiogenesis³⁷.Artificial cerebrospinal fluid (aCSF), infused as control, was also welltolerated for prolonged periods, indicating that the surgical procedureswere safe. Another advantage of this low VEGF dose was that it did notinduce an immune response. Indeed, no anti-VEGF antibodies weredetectable in the peripheral blood or CSF of rats, not even after 100days of delivery of a dose range of 0.2 μg/kg/day to 0.6 μg/kg/day ofVEGF.

4. Effect of VEGF in a Rat Model of ALS

SOD1^(G93A) rats have not been previously used for evaluating novel ALStreatment paradigms. We used SOD1^(G93A/LSd) rats, generated by Nagai etal¹⁰ (“L” refers to the low, e.g. 2-fold increased, SOD1^(G93A) levelsin this model) on a Sprague-Dawley background (Sd). Disease progressionis very aggressive in this model, killing the animals within 10 daysafter disease onset¹⁰, SOD1^(G93A/LSd) rats exhibited a largeinter-litter variability in disease onset, ranging from 95 to 145 days.To reduce this variability, we used SOD1^(G93A/LSd) littermates andanalyzed the results in a paired manner (N=17 rats analyzed in 6litter-pairs; see methods for details). Compared to control artificialCSF (aCSF), treatment of SOD1^(G93A/LSd) rats with 0.6 μg VEGF/kg/day at60 days of age significantly delayed disease onset, and improved motorperformance and overall clinical outcome, regardless of the scoringmethod. For instance, VEGF-treated SOD1^(G93A/LSd) rats remainedgenerally active, mobile, attentive, and groomed their fur, at a timewhen aCSF-animals already showed signs of paralysis, and wereprogressively becoming immobile and cachectic. Control animals had moresevere muscle atrophy than VEGF-treated rats. ICV delivery of VEGFdelayed, by 10 days, the onset of limb paralysis, scored as dragging ofa hindlimb or failure to use a forelimb during walking or righting(P<0.05). Using a laser-beam detection system¹⁰, we identified the ageat which rats were no longer capable of crossing equally spacedlaser-beams in an “activity cage” at least 1,000 times per hour—ameasure of their spontaneous walking behaviour. After VEGF delivery,rats remained spontaneously active at an older age (135±5 days, aCSFversus 146±8 days, VEGF; P<0.05). We also videotaped the rats anddetermined the time the rats spent exploring their cage and counted thefrequency the rats reared themselves upon their hindlimbs as additionalmeasures of their spontaneous activity. Before disease onset, i.e. at110 days of age, both groups explored their cage as actively and rearedthemselves as frequently (P=NS). Four days after aCSF-rats showed thefirst signs of limb paralysis, VEGF-treated rats explored their cageslonger and reared themselves more frequently than aCSF-treated animals(P<0.05). Finally, VEGF prolonged the survival of these ALS rats by 10days (P<0.01). Thus, despite the very rapid disease progression and thelarge inter-litter variability of disease onset in this model,VEGF-treatment delayed onset, improved motor performance and prolongedsurvival of SOD1^(G93A/LSd) rats without causing adverse effects.

5. Molecular Mechanism of the Neuroprotective Effect of VEGF

We further explored the mechanisms by which VEGF prolonged motoneuronsurvival in vivo. In vitro studies indicated that VEGF protectsmotoneurons against stress-induced cell death by binding VEGF receptor-2(also termed Flk1)¹³, but a direct neurotrophic activity of VEGF in vivohas never been demonstrated. To address the latter issue, we generatedtransgenic mice, using the Thy1.2 expression cassette to driveexpression of murine Flk1 in postnatal neurons⁴⁵. Compared tonon-transgenic littermates, Thy-Flk1 mice expressed more Flk1 mRNAtranscripts (copies Flk1/10³ copies HPRT: 470±45 versus 50±5; N=3;P<0.05) and protein. Flk1 expression in non-transgenic littermates wasdetectable in blood vessels and, at a lower level, in large motoneurons.In contrast, in Thy-Flk1 mice, high Flk1 levels were present on largemotoneurons in the ventral horn, in addition to its baseline expressionin endothelial cells. Thy-Flk1 mice appeared healthy and fertile andwere intercrossed with SOD1^(G93A) mice. Notably, neuronaloverexpression of Flk1 in SOD1^(G93A) mice delayed onset of motorimpairment by 23 days (N=8; P<0.001) and Thy1-Flk1:SOD1^(G93A) miceperformed better than SOD1^(G93A) mice during 26 days (N=8; P<0.01). Inaddition, Thy1-Flk1:SOD1^(G93A) mice survived 10 days longer than theirSOD1^(G93A) littermates (N=8, P<0.05). Thus, these genetic findingsindicate that Flk1 on motoneurons transmits key survival signals ofendogenous VEGF, thereby delaying premature motoneuron degeneration inALS. Further evidence for a neuroprotective effect of Flk1 was providedby generating transgenic mice, using the same Thy1.2 expression cassetteto drive neuronal expression of a dominant-negative Flk1, which impairsVEGF signaling (Flk1^(DN)). Thy-Flk1^(DN) mice also expressed elevatedlevels of the Flk1^(DN) transgene in motoneurons. At 3 months of age,Thy-Flk1^(DN) mice were healthy and fertile, and had normal musclestrength, motor performance and numbers of motoneurons (SMI32⁺motoneurons/ventral horn: 31±4.4, wild type mice versus 29±1.2,Thy-Flk1^(DN) mice; N=4; P=NS). To stress the motoneurons, the mice werehoused, every other day for 30 days, in a chamber with 10% O₂. Chronicintermittent hypoxia upregulated VEGF levels in the spinal cord (pgVEGF/μg protein: 12±0.5, normoxia versus 22±1.4, hypoxia; N=5; P<0.05).Wild type mice tolerated the hypoxia without any problem and their gripstrength even slightly increased. In contrast, Thy-Flk1^(DN) mice lost25% of their grip strength within one week after exposure to hypoxia andremained weaker for the rest of the experiment. Histological analysisrevealed a marked gliosis in the gray matter of Thy-Flk1^(DN) mice butnot in wild type mice (GFAP⁺ area/gray matter area in ventral horn:0.13±0.45%, wild type versus 2.7±0.45%, Thy-Flk1^(DN); N=3-5; P<0.05).Furthermore, motoneurons in Thy-Flk1^(DN) but not in wild type miceaccumulated phosphorylated neurofilament (SMI31⁺ neurons/10 ventral hornsections: none in wild type versus 15.3±7.2 in Thy-Flk1^(DN); N=3-5;P<0.05). These findings thus illustrate that Flk1 has a criticalprotective role in adult motoneuron maintenance in conditions of hypoxicstress.

Materials and Methods

1. Production of recombinant rat VEGF₁₆₄ (VEGF₁₆₄).

VEGF₁₆₄ cDNA amplified from a rat cDNA library was cloned into thepPICZαA secretion vector and expressed using the Pichia pastoris yeastexpression system, according to the instructions of the manufacturer(Invitrogen, Carlsbad, Calif.). After overnight dialysis of theyeast-conditioned medium against 10 mM acetic acid (pH 5.5), VEGF waspurified by sequential chromatography on phenyl Sepharose 6 Phast flowand heparin-agarose columns (both from Amersham Pharmacia Biotech). TheVEGF concentration was determined using the rat Duoset ELISA (R&DSystems, Abingdon, UK). The purified VEGF₁₆₄ was then electrophoresedand single 45 kDa or 22 kDa-stained bands under non-reducing andreducing conditions, respectively, were visualized by coomassie blue-and silver-staining of the gels. The band was confirmed to berecombinant rat VEGF₁₆₄ by immunoblotting with a monoclonal antibodyspecific for rVEGF₁₆₄ (R&D Systems) and N-terminal sequencing afterEdman's degradation protocol. After trypsin digestion of the prominent45 kDa band, which was followed by separation of the cleaved peptides ondHPLC, 3 internal peptides were selected and N-terminally sequencedaccording to Edman's degradation protocol. All three peptides were aperfect match to the published VEGF164 amino acid sequence.

2. Functional Characterization of VEGF₁₆₄

Binding of our VEGF₁₆₄ preparation to immobilized rhFc-FLT1 andrhFc-FLK1 (R&D Systems) receptors was compared with that of commerciallyavailable rat VEGF₁₆₄. Bound VEGF₁₆₄ was detected using biotinylatedanti-rat VEGF antibodies (R&D Systems; 200 ng/ml), the ABC vectorstaining kit and photospectrometric readout. Home-made VEGF₁₆₄ exhibiteda higher affinity towards rhFc-FLK1 and a similar binding affinitytowards rhFc-FLT1. Endotoxin levels were determined by using the LimulusAmebocyte Lysate (LAL) kit (Bio-Whittaker, Walkersville, USA) andrevealed the presence of 1 endotoxin unit per 350 μg VEGF₁₆₄ (or 3 E10-3endotoxin unit per μg VEGF₁₆₄).

3. Radio-Labeled Experiments

Radio-labeled human ¹²⁵I-VEGF was purchased from Amersham Pharmacia witha specific activity of 25 μCi/μg VEGF₁₆₅. 100 ng of this preparation wasdissolved in 10 μL and stereotactically injected into the left lateralventricle of healthy female Wistar rats by using a 33 Gauge Hamiltonneedle—stereotactic coordinates were the same as for the implantation ofthe osmotic pump (see below). Subsequent to ICV-injection, rats weredissected after 1, 3 and 24 hours, and the brain, spinal cord (dividedinto cervical, thoracal and lumbal spinal cord), blood and other organs(liver, intestine, heart, etc) weighed and the total counts per minute(cpm) measured using a gamma-counter. The distribution of ¹²⁵I-hVEGF₁₆₅in the parenchyma of the brain was evaluated by microautoradiography.First, cryosections of the brain and spinal cord, containing¹²⁵I-labeled VEGF₁₆₅ were dipped into photographic emulsion (Kodak,Cedex, France). After two days of exposure, the emulsified sections weredeveloped and the location of silver grains, as detected by lightmicroscopy, was used to determine the tissue distribution of the¹²⁵I-labeled VEGF₁₆₅.

4. Generation and Characterization of Transgenic Mice

Transgenic mice expressing Flk-1 specifically in adult neurons weregenerated using the mouse Thy1.2 expression cassette, as previouslydescribed 59. Murine Flk-1 cDNA was cloned into the Thy-1.2 expressioncassette, and the linearized construct was microinjected into FvB mouseembryos using standard microinjection techniques. Founders wereidentified using PCR and expression of the transgene was determined byRT-PCR, Western blotting and immunostaining, as previouslydescribed^(13,37).

5. Animals

Sprague-Dawley rats expressing the human SOD1^(G93A) transgene(SOD1^(G93A)-L) were kindly provided by Dr. Itoyama¹⁰, while miceexpressing the human SOD1^(G93A) transgene were backcrossed for morethan 10 generations on a FvB background and were kindly provided by Dr.C. Kunst⁶⁰. All experiments on animals were approved by the local animalethics committee.

6. Surgical Procedures

To infuse recombinant rat VEGF₁₆₄ into the cerebral ventricle of therats, Alzet osmotic pumps (model 2004) connected with a catheter to abrain infusion cannula were used. The brain infusion assembly was filledwith 200 μl of a 5 μg/ml recombinant rat VEGF₁₆₄ solution or withartificial CSF, and primed during 48 h in saline. The composition of theartificial CSF was 150 mM Na⁺, 3 mM K⁺, 1.4 mM Ca²⁺, 0.8 mM Mg²⁺, 1 mMPO₄ ³⁻, 155 mM Cl⁻. For the implantation of the pumps, rats wereanesthetized with halothane, a midline sagittal incision was madestarting slightly behind the eyes, and the skull was exposed. Asubcutaneous pocket in the midscapular area of the back of the rat wasprepared, and the osmotic pump was inserted into the pocket. A hole wasdrilled through the skull, and the cannula placed using the followingstereotaxic coordinates: 0.8 mm posterior to bregma, 1.6 mm lateral, and4.5 mm ventral from skull surface. When the implantation procedure wascompleted, the skin incision was sutured, and the rat allowed torecover. After 28 days, the emptied osmotic pumps were replaced by afresh, fully loaded and primed osmotic pump. To do so, the rat wasanesthetized again and a small skin incision in the midscapular regionof the back was made. The catheter was cut 5 mm anterior to the spentpump, and a fresh pump was attached to the catheter tubing. Thisprocedure results in a continuous infusion of VEGF₁₆₄, at a rate of 0.25μl/h (corresponding to 1.25 ng VEGF/hour) into the CSF. In a preventiontrial, pumps were implanted at the age of 60 days, and in a regressiontrial, pumps were implanted at 80 days (age of disease onset). Mice wereexposed to chronic intermittent hypoxia by transferring them, everyother day during 30 days, to an oxygen chamber, containing 12% oxygen.

7. Behavioural Analysis

Three times a week, motor performance of the rats was tested usingrotarod, dynamometer, and spontaneous activity measurements. A rotarodfor rats (Ugo Basile, Comerio VA, Italy) with a constant rotation speed(15 rotations per minute) was used. The average of 5 trials of maximum180 seconds was determined on a given day. When the average time a ratcould stay on the rotarod was less than 120 seconds, this was considereda failure. For the dynamometer test, an average of 5 trials was made foreach rat, and when the rat was unable to pull on average 800 mg, thiswas considered a failure. To quantify spontaneous activity, rats wereplaced for 3 hours into an activity cage (Ugo Basile), and the averageactivity per hour, i.e. the number of times the rat crossed an infraredbeam positioned 10 cm above the cage floor, was calculated. As acriterion to score the age of disease onset of the rats, dragging of onelimb during walking was used. In previous studies, failure of the animalto right itself after being turned on either side for a maximum of 30seconds was scored as “clinical death”⁹. However, initial experimentsrevealed that SOD1^(G93A) rats, unable to right themselves, could stillsurvive for several days—this was particularly the case for rats withforelimb disease. We therefore scored the time of death as the day therats had lost 40% of their original body weight at the lastpresymptomatic age, as experiments indicated that the animals within24-36 hours thereafter. The data generated by the described clinicaltests and the survival data were analyzed using Kaplan Meyer statisticalanalysis or by using ANOVA repeated measures (rotarod, dynamometer andactivity).

8. Histology, Immunohistochemistry and ELISAs

Animals were perfused transcardially, under deep Nembutal anaesthesia,with 0.9% NaCl solution followed by 1% phosphate bufferedparaformaldehyde. Spinal cord and brain were dissected, post-fixed inthe same fixative overnight, dehydrated and embedded in paraffin. Serialsections were cut at 20 μm thickness for the brain and at 7 μm for thespinal cord. For immunohistochemistry, primary antibodies were used asfollows: mouse anti-SMI-32 and mouse anti-SMI-31 (both 1:500,Sternberger Monoclonals); mouse anti-GFAP (1:400, Sigma); goatanti-Glut-1 (1:20, Santa Cruz Biotechnology); rabbit anti-ubiquitin(1/100, Dako) and rabbit anti-albumin (1:250, ICN/Cappel). Formotoneuron counts in the spinal cord, SMI-32 positive neurons in theventral horn were counted bilaterally on 5 equally spaced sections overa distance of 350 μm. To determine the number of motoneurons in thefacial nucleus, every 10^(th) brain stem section was stained, and allSMI-32 positive neurons in de facial nucleus region counted. This numberwas multiplied by ten, to estimate the total number of motoneurons inthe facial nucleus.

9. Immune Response

Levels of VEGF₁₆₄ in the spinal cord and in the plasma were below theELISA detection limit (32.5 pg/mL; R&D Systems) in both aCSF andVEGF-treated mice (n=7 for each group). To detect whether there werecirculating anti-VEGF antibodies present in VEGF-treated rats, 96-wellmicrotiter plates were coated overnight with 100 μl of a 1 μg/mlsolution of VEGF₁₆₄ protein. After incubation with plasma or CSF fromaCSF- and VEGF-treated rats, bound anti-VEGF₁₆₄ antibodies were detectedby using HRP-labelled anti-rat immunoglobulins (DAKO; 200 ng/ml), theABC vector staining kit and photospectrometric readout.

10. Statistics

We used SPSS version 10 for all statistical calculations. Cumulativesurvival statistics were calculated by using Kaplan-Meier statistics.The spontaneous activity, rotarod and weight loss data were analyzedusing repeated-measures ANOVA. Student t-tests were used to calculatesignificant differences for the histological studies.

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1. A method for treating amyotrophic lateral sclerosis (ALS) orenhancing the survival of a motor neuron in a subject diagnosed with amotoneuron disease, said method comprising: administration of a vascularendothelial growth factor-A (“VEGF-A”) protein into the cerebrospinalfluid of the subject at a dose within a range of 0.05 μg/kg/day and 1μg/kg/day of the subject's body mass, wherein the VEGF-A protein isselected from the group consisting of: VEGF₁₂₁, VEGF₁₄₅, and VEGF₁₆₅,and VEGF₁₈₉, thus treating the ALS disease or enhancing the survival ofa motor neuron in the subject diagnosed with a motoneuron disease. 2.The method according to claim 1 wherein said dose is administered withina range of 0.2 μg/kg/day and 0.8 μg/kg/day.
 3. The method according toclaim 1 wherein said administration is intrathecal.
 4. The methodaccording to claim 1 wherein said administration isintracerebroventricular.
 5. The method according to claim 1 wherein saidcontinuous administration of VEGF occurs via an implanted osmoticmini-pump.
 6. A method for treating amyotrophic lateral sclerosis (ALS)or enhancing the survival of a motor neuron in a subject diagnosed witha motoneuron disease, the method comprising: administering a vascularendothelial growth factor (“VEGF”) protein to the subject at a dosagerange of between 0.05 μg/kg/day and 1 μg/kg/day of the subject's bodymass, wherein the VEGF protein is selected from the group consisting ofVEGF₁₂₁, VEGF₁₄₅, and VEGF₁₆₅, and VEGF₁₈₉, and wherein theadministration is intrathecal or intracerebroventricular, thus treatingthe ALS disease or enhancing the survival of a motor neuron in thesubject diagnosed with a motoneuron disease.
 7. A method for treatingamyotrophic lateral sclerosis (ALS) or enhancing the survival of a motorneuron in a subject diagnosed with a motoneuron disease, the methodcomprising: administering a vascular endothelial growth factor (“VEGF”)protein into the cerebrospinal fluid of the subject at a dosage range ofbetween 0.05 μg/kg/day and 1 μg/kg/day of the subject's body mass,wherein the VEGF protein is selected from the group consisting ofVEGF₁₂₁, VEGF₁₄₅, and VEGF₁₆₅, and VEGF₁₈₉, thus treating the ALSdisease or enhancing the survival of a motor neuron in the subjectdiagnosed with a motoneuron disease.